Downregulation of c-Met expression does not enhance the sensitivity of gastric cancer cell line MKN-45 to gefitinib

Ma,Jin‑An; Hu,Chunhong; Li,Wenjuan; Ren,Jing; Zou,Fangwen; Zhou,Dongai; Zou,Wen; Wei,Yajun; Zhou,Ying

doi:10.3892/mmr.2014.2948

Downregulation of c-Met expression does not enhance the sensitivity of gastric cancer cell line MKN-45 to gefitinib

Authors:
- Jin‑An Ma
- Chunhong Hu
- Wenjuan Li
- Jing Ren
- Fangwen Zou
- Dongai Zhou
- Wen Zou
- Yajun Wei
- Ying Zhou
View Affiliations

Affiliations: Department of Oncology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, P.R. China
Published online on: November 13, 2014 https://doi.org/10.3892/mmr.2014.2948
Pages: 2269-2275

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The aim of the present study was to investigate the effect of downregulation of the c‑Met gene on signal transduction and apoptosis in gastric cancer MKN‑45 cells; furthermore, the study aimed to determine whether altered c‑Met gene expression affected MKN‑45 sensitivity to gefitinib. Three c‑Met‑specific small interfering RNAs (siRNAs) were synthesized and transfected into MKN‑45 cells. Messenger RNA (mRNA) and protein levels of c‑Met and its downstream signaling molecules [phosphoinositide 3‑kinase (PI3K) and AKT] were examined using reverse transcription polymerase chain reaction and western blot analysis 48 h following transfection. Cell apoptosis was evaluated using Annexin‑V/propidium iodide double staining and fluorescence‑activated cell sorting analysis. An MTT assay was performed in order to measure the 50% inhibitory concentration (IC50) of gefitinib on MKN‑45 cells. The results of the present study demonstrated that 48 h post‑transfection with c‑Met siRNA, MKN‑45 cells showed significantly downregulated expression of c‑Met mRNA and protein as well as an increased rate of apoptosis (P<0.05). In addition, following c‑Met siRNA transfection mRNA and protein levels of PI3K and AKT were not significantly altered in MKN‑45 cells (P>0.05); however, a marked decrease in the expression levels of phosphorylated (p)‑PI3K and p‑AKT was observed (P<0.05). Furthermore, the IC50 of gefitinib in MKN‑45 cells was not significantly decreased. In conclusion, knockdown of the c‑Met gene promoted gastric cancer cell apoptosis and inhibited downstream p‑PI3K and p‑AKT; however, the sensitivity of MKN‑45 cells to gefitinib was not increased.

View Figures

View References

1	Lordick F and Siewert JR: Recent advances in multimodal treatment for gastric cancer: a review. Gastric Cancer. 8:78–85. 2005. View Article : Google Scholar : PubMed/NCBI
2	Bang YJ, Van Cutsem E, Feyereislova A, et al: Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet. 376:687–697. 2010. View Article : Google Scholar : PubMed/NCBI
3	Zhang J, Liu H, Zhu R, Hinterdorfer P, Zhang B and Tang J: Single molecular dissection of the ligand binding property of epidermal growth factor receptor. Analyst. 138:5325–5331. 2013. View Article : Google Scholar : PubMed/NCBI
4	Liakakos T, Xeropotamos N, Ziogas D and Roukos D: EGFR as a prognostic marker for gastric cancer. World J Surg. 32:1225–1226. 2008. View Article : Google Scholar : PubMed/NCBI
5	Janjigian YY, Tang LH, Coit DG, et al: MET expression and amplification in patients with localized gastric cancer. Cancer Epidemiol Biomarkers Prev. 20:1021–1027. 2011. View Article : Google Scholar : PubMed/NCBI
6	Huang TJ, Wang JY, Lin SR, Lian ST and Hsieh JS: Overexpression of the c-met protooncogene in human gastric carcinoma - correlation to clinical features. Acta Oncol. 40:638–643. 2001. View Article : Google Scholar
7	Drebber U, Baldus SE, Nolden B, et al: The overexpression of c-met as a prognostic indicator for gastric carcinoma compared to p53 and p21 nuclear accumulation. Oncol Rep. 19:1477–1483. 2008.PubMed/NCBI
8	Amemiya H, Menolascino F and Peña A: Role of the expression of c-Met receptor in the progression of gastric cancer. Invest Clin. 51:369–380. 2010.(In Spanish).
9	Namiki Y, Namiki T, Yoshida H, et al: Preclinical study of a ‘tailor-made’ combination of NK4-expressing gene therapy and gefitinib (ZD1839, Iressa) for disseminated peritoneal scirrhous gastric cancer. Int J Cancer. 118:1545–1555. 2006. View Article : Google Scholar
10	Rojo F, Tabernero J, Albanell J, et al: Pharmacodynamic studies of gefitinib in tumor biopsy specimens from patients with advanced gastric carcinoma. J Clin Oncol. 24:4309–4316. 2006. View Article : Google Scholar : PubMed/NCBI
11	Dragovich T, McCoy S, Fenoglio-Preiser CM, et al: Phase II trial of erlotinib in gastroesophageal junction and gastric adenocarcinomas: SWOG 0127. J Clin Oncol. 24:4922–4927. 2006. View Article : Google Scholar : PubMed/NCBI
12	Zheng Y, Asara JM and Tyner AL: Protein-tyrosine kinase 6 promotes peripheral adhesion complex formation and cell migration by phosphorylating p130 CRK-associated substrate. J Biol Chem. 287:148–158. 2012. View Article : Google Scholar :
13	Chell V, Balmanno K, Little AS, et al: Tumour cell responses to new fibroblast growth factor receptor tyrosine kinase inhibitors and identification of a gatekeeper mutation in FGFR3 as a mechanism of acquired resistance. Oncogene. 32:3059–3070. 2013. View Article : Google Scholar
14	Tian W, Chen J, He H and Deng Y: MicroRNAs and drug resistance of breast cancer: basic evidence and clinical applications. Clin Transl Oncol. 15:335–342. 2013. View Article : Google Scholar
15	Karamouzis MV, Konstantinopoulos PA and Papavassiliou AG: Targeting MET as a strategy to overcome crosstalk-related resistance to EGFR inhibitors. Lancet Oncol. 10:709–717. 2009. View Article : Google Scholar : PubMed/NCBI
16	Onitsuka T, Uramoto H, Nose N, et al: Acquired resistance to gefitinib: the contribution of mechanisms other than the T790M, MET, and HGF status. Lung Cancer. 68:198–203. 2010. View Article : Google Scholar
17	Bean J, Brennan C, Shih JY, et al: MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci USA. 104:20932–20937. 2007. View Article : Google Scholar : PubMed/NCBI
18	Cao WG, Ma T, Li JF, et al: Effect of gefitinib on radiosensitivity of gastric cancer cell lines. Chinese Journal of Cancer. 26:1330–1335. 2007.(In Chinese).
19	Fushida S, Yonemura Y, Urano T, et al: Expression of hepatocyte growth factor(hgf) and C-met gene in human gastric-cancer cell-lines. Int J Oncol. 3:1067–1070. 1993.PubMed/NCBI
20	Casagrande G, te Kronnie G and Basso G: The effects of siRNA-mediated inhibition of E2A-PBX1 on EB-1 and Wnt16b expression in the 697 pre-B leukemia cell line. Haematologica. 91:765–771. 2006.PubMed/NCBI
21	Li Y, Huang X, Zhong W, Zhang J and Ma K: Ganglioside GM3 promotes HGF-stimulated motility of murine hepatoma cell through enhanced phosphorylation of cMet at specific tyrosine sites and PI3K/Akt-mediated migration signaling. Mol Cell Biochem. 382:83–92. 2013. View Article : Google Scholar : PubMed/NCBI
22	Kao J, Sikora AT and Fu S: Dual EGFR and COX-2 inhibition as a novel approach to targeting head and neck squamous cell carcinoma. Curr Cancer Drug Targets. 9:931–937. 2009. View Article : Google Scholar : PubMed/NCBI
23	Chen HJ, Mok TS, Chen ZH, et al: Clinicopathologic and molecular features of epidermal growth factor receptor T790M mutation and c-MET amplification in tyrosine kinase inhibitor-resistant Chinese non-small cell lung cancer. Pathol Oncol Res. 15:651–658. 2009. View Article : Google Scholar : PubMed/NCBI
24	Rosenzweig SA: Acquired resistance to drugs targeting receptor tyrosine kinases. Biochem Pharmacol. 83:1041–1048. 2012. View Article : Google Scholar : PubMed/NCBI
25	Agarwal S, Zerillo C, Kolmakova J, et al: Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells. Br J Cancer. 100:941–949. 2009. View Article : Google Scholar : PubMed/NCBI
26	Kosaka T, Yamaki E, Mogi A and Kuwano H: Mechanisms of resistance to EGFR TKIs and development of a new generation of drugs in non-small-cell lung cancer. J Biomed Biotechnol. 2011:1652142011. View Article : Google Scholar : PubMed/NCBI
27	Engelman JA, Zejnullahu K, Mitsudomi T, et al: MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039–1043. 2007. View Article : Google Scholar : PubMed/NCBI
28	Que W and Chen J: Knockdown of c-Met inhibits cell proliferation and invasion and increases chemosensitivity to doxorubicin in human multiple myeloma U266 cells in vitro. Mol Med Rep. 4:343–349. 2011.PubMed/NCBI
29	Wang ZX, Lu BB, Yang JS, Wang KM and De W: Adenovirus-mediated siRNA targeting c-Met inhibits proliferation and invasion of small-cell lung cancer (SCLC) cells. J Surg Res. 171:127–135. 2011. View Article : Google Scholar
30	Xie B, Xing R, Chen P, et al: Down-regulation of c-Met expression inhibits human HCC cells growth and invasion by RNA interference. J Surg Res. 162:231–238. 2010. View Article : Google Scholar
31	Shinomiya N, Gao CF, Xie Q, et al: RNA interference reveals that ligand-independent met activity is required for tumor cell signaling and survival. Cancer Res. 64:7962–7970. 2004. View Article : Google Scholar : PubMed/NCBI
32	Teng L and Lu J: cMET as a potential therapeutic target in gastric cancer (Review). Int J Mol Med. 32:1247–1254. 2013.PubMed/NCBI
33	Wheeler DL, Huang S, Kruser TJ, et al: Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members. Oncogene. 27:3944–3956. 2008. View Article : Google Scholar : PubMed/NCBI
34	Sequist LV, von Pawel J, Garmey EG, et al: Randomized phase II study of erlotinib plus tivantinib versus erlotinib plus placebo in previously treated non-small-cell lung cancer. J Clin Oncol. 29:3307–3315. 2011. View Article : Google Scholar : PubMed/NCBI